WO2019114374A1 - 一种乳制品中沙门氏菌的快速检测方法 - Google Patents

一种乳制品中沙门氏菌的快速检测方法 Download PDF

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WO2019114374A1
WO2019114374A1 PCT/CN2018/108873 CN2018108873W WO2019114374A1 WO 2019114374 A1 WO2019114374 A1 WO 2019114374A1 CN 2018108873 W CN2018108873 W CN 2018108873W WO 2019114374 A1 WO2019114374 A1 WO 2019114374A1
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primer
salmonella
dairy product
detection
reaction solution
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王文文
张海涛
史寒琴
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新希望双喜乳业（苏州）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to microbial testing, in particular to a rapid detection method of Salmonella in dairy products.
  • Dairy products are rich in nutrients and balanced, and are an economical and high-quality protein.
  • China is a big consumer of dairy products and the third largest dairy producer in the world.
  • dairy products are highly susceptible to pathogen contamination.
  • Common food-borne pathogens that contaminate dairy products include Salmonella, Staphylococcus aureus, Enterobacter sakazakii, etc., which cause diarrhea, vomiting, and acute after entering the human body. Gastroenteritis and other diseases can seriously endanger life.
  • Salmonella and Staphylococcus aureus are the most common items for dairy detection.
  • Salmonella is a common food-borne pathogen in raw milk and dairy products. People and animals infected with it may develop typhoid fever and paratyphoid fever. Once Salmonella enters blood tissue, it may cause systemic inflammation and even death. .
  • Salmonella testing is required for raw materials, process products, final products, and production environments during processing. According to GB4789.1-2016, the same batch needs to collect 5 samples for testing, and the detection amount is up to several hundred per day. According to the requirements of GB4789.4-2016, the qualitative determination of Salmonella requires at least 3 days. This method is cumbersome and time consuming, and cannot meet the requirements of testing aging.
  • pretreatment adding the dairy product to be tested to a solution containing sodium diacetamide tetraacetate and glucoside, and boiling in a water bath at 40-42 ° C for 50-60 minutes;
  • the FIP primer, the BIP primer, the F3 primer and the B3 primer are respectively a pre-inner primer, a post-primer primer, a pre-external primer and a post-primer primer in a LAMP detection primer of the Salmonella invA gene;
  • step (3) the amplification reaction solution prepared in step (3) is reacted at 65 ° C for 45-50 minutes to perform fluorescence color detection; when the LAMP amplification reaction solution changes from brown to yellow-green fluorescence , with positive amplification; when the LAMP amplification reaction solution has no color change, there is no positive amplification.
  • the concentration of sodium diacetamide tetraacetate in the solution described in the step (1) is 40-50% by weight, and the mass concentration of the glucoside is 20-25% by weight.
  • the mass ratio of the dairy product, sodium diacetamide tetraacetate, and glucoside in step (1) is 1:4-5:2-2.5.
  • the filtration in step (2) is performed aseptically.
  • the centrifugation described in step (2) is performed by centrifugation at 12000 rpm for 5 minutes.
  • the sequence composition of the FIP primer is 5'-GCGCCGCTA CGCACTCTA TATGTAGCT GCTACGCAC GG-3';
  • the sequence composition of the BIP primer is 5'-GAACGGTGA AGCTTACGG GACGTCACT CGGTCATAG GAA-3';
  • the sequence composition of the F3 primer is 5'-GAACATGCT GCAGATGTC-3';
  • the sequence composition of the B3 primer was 5'-CGGCATTAG CGACTCCTT-3'.
  • the fluorescent color detection in the step (4) employs an IQ TN 5 fluorescence quantitative PCR machine optical system.
  • the method for detecting Salmonella in dairy products according to the invention is simple, time-saving, high-accuracy and high-sensitivity, and can only be required from the pretreatment of the dairy product to the final detection result. 3.5 hours, greatly improving the freshness and shelf life of the tested dairy products, the positive result of the method detected by the method of the present invention and the result detected according to the steps in GB4789.4-2016 reached 99.9% or more.
  • the dairy product to be tested is pretreated with a solution containing sodium diacetamide tetraacetate (EDTTI-Na) and glucoside (APG), and the structure of EDTTI-Na is similar to EDTA, and substituted with an amide group.
  • EDTTI-Na sodium diacetamide tetraacetate
  • APG glucoside
  • EDTTI-Na is used in the assisted action of APG to capture dairy casein micelles.
  • Calcium ion a macromolecular protein polymer in the decomposition system, dissociates the casein micelles into small molecule casein monomers, dissociates the protein polymer into short peptide chains, overcomes protein micelles or polymers in a vacuum
  • the repression in the enrichment process is filtered, so that the sample to be detected, that is, the bacteria containing Salmonella or the like can be rapidly enriched.
  • Salmonella can be effectively amplified; (2) Fluorescent color reaction reagent, eosin and 2-hydroxy-3-naphthoic acid are added to the reaction solution, and the two are not specific for the amplification product of Salmonella target gene when used alone.
  • the sexual fluorescent color development effect when added according to the ratio according to the present invention, has a significant color distinction between the negative and positive reaction of the Salmonella invA gene, and the result can be accurately judged according to this, that is, when the LAMP amplification reaction solution changes from brown to brown When the yellow-green fluorescence is positive, it means positive amplification, which means that there is Salmonella contamination in the sample; when there is no color change in the LAMP amplification reaction solution, there is no positive amplification, indicating that there is no Salmonella in the sample. Dye.
  • the fluorescence detection of Salmonella has high sensitivity, and the detection sensitivity of the commonly used PCR electrophoresis is improved by more than 10 times, and the operation is simple, and the enrichment efficiency of the target gene is high, which is sufficient in the half-hour amplification time. The limit that can be detected.
  • the present invention will be further described in detail below with reference to the embodiments, so that those skilled in the art can refer to the description.
  • the present invention provides a rapid detection method for Salmonella in dairy products, which is specifically illustrated by the following examples.
  • pretreatment 1 part by weight of the milk to be tested is added to 10 parts by weight of a solution containing 50% by weight of sodium diacetamide tetraacetate and 25 wt% glucoside, and water bath at 40-42 ° C for 50-60 minutes;
  • the mixture solution after the water bath in the step (1) is subjected to sterile vacuum filtration, and the bacteria on the filter membrane are eluted with physiological saline, and then the eluate is centrifuged at 12,000 rpm for 5 minutes, and discarded. The supernatant was removed, 1.2 mL of TE buffer solution was added to the precipitate, and the precipitate was suspended by shaking, centrifuged again, and the supernatant was discarded, and the DNA in the test sample was quickly extracted by the bacterial genomic DNA extraction kit;
  • the amplification reaction solution prepared in the step (3) is reacted at 65 ° C for 45 minutes, and the fluorescent color detection is performed by an IQ TN 5 fluorescence quantitative PCR optical system; when the LAMP amplification reaction solution is used When it changed from brown to yellow-green fluorescence, it showed positive amplification, indicating the presence of Salmonella in the sample. When there was no color change in the LAMP amplification reaction solution, there was no positive amplification, indicating that there was no Salmonella in the sample.
  • the first part can detect whether the milk is contaminated by Salmonella by the method in Example 1, and the result can be obtained within 3 hours.
  • the second part is carried out according to the steps in GB4789.4-2016.
  • Salmonella detection it takes up to 3 working days to get the test results; in no less than 100 tests, the two methods are completely consistent with the Salmonella test results.
  • the detection time by the Salmonella test method of the present invention is about 3.5 hours, and in the test of not less than 100 times, the result is the same as that passed the test by GB4789.4-2016.
  • Example 1 The sodium diacetamide tetraacetate and the glucoside in the step (1) of Example 1 were replaced with the same concentration of the conventional reagent oxalic acid tetraacetic acid (EDTA) to carry out pretreatment of the dairy product, and the remaining detection methods were the same as in the examples.
  • EDTA conventional reagent oxalic acid tetraacetic acid
  • step (2) After replacement with EDTA, the resistance in the vacuum enrichment of bacteria in step (2) is greater, the repression effect is obvious, and the enrichment time of the equal sample is increased by about 25-30 minutes.
  • the results of fluorescent color detection showed that the five different serotypes of Salmonella LAMP reaction solution changed from brown to yellow-green fluorescence, indicating positive amplification.
  • the LAMP reaction solution was brown before and after the negative control reaction, no color change, indicating no positive expansion. increase.
  • Salmonella genomic DNA Five Enterobacteriaceae (Serratia, Proteus, Shigella, Escherichia coli, Klebsiella) similar to the physiological and biochemical characteristics of Salmonella were selected for extraction of genomic DNA by boiling method, and Salmonella genomic DNA was used.
  • the positive control, double distilled water, and the negative control were subjected to LAMP amplification and detection according to the methods in the steps (3) and (4) of Example 1.
  • the genomic DNA of S. Enteritidis standard strain was extracted by boiling method and diluted 10-fold to obtain concentrations of 445 ng/ ⁇ L, 44.5 ng/ ⁇ L, 4.45 ng/ ⁇ L, 445 pg/ ⁇ L, 44.5 pg/ ⁇ L, 4.45 pg/ ⁇ L, 0.445. Pg/ ⁇ L, 0.0445 pg/ ⁇ L of S. Enteritidis genomic DNA.
  • Different gradient DNA concentrations were subjected to LAMP amplification and detection according to the methods in the steps (3) and (4) of Example 1.
  • the LAMP reaction solution of S. enteritid After 45 minutes of amplification reaction, the LAMP reaction solution of S. enteritidis changed from 445ng/ ⁇ L to 0.445pg/ ⁇ L to yellow-green fluorescence, and the LAMP reaction solution did not appear at a concentration of 0.0442pg/ul. The color changes to brown.
  • the minimum of the detection line is more than 10 times lower than the detection limit in the prior art, and the sensitivity to Salmonella is greatly improved.

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Abstract

一种乳制品中沙门氏菌的快速检测方法，主要包括对乳制品的预处理、提取待测样品中DNA模板、配制LAMP扩增反应液、LAMP扩增及检测等步骤；其中在LAMP扩增反应液中加入了曙红和2-羟基-3-萘甲酸，使得阳性扩增产物表现出特异的荧光反应，从而确定了沙门氏菌的存在；这种对乳制品中沙门氏菌的检测方法简单省时、准确度高、灵敏度高，从对乳制品的预处理到得到最终检测结果能够仅需要3-3.5小时，极大的提高了被检乳制品的新鲜度和货架期，通过本方法检测出的结果与按照GB 4789.4-2016中步骤检测出的结果的C12Q阳性符合率达到99.9％以上。

Description

一种乳制品中沙门氏菌的快速检测方法 技术领域
本发明涉及微生物检验，具体涉及一种乳制品中沙门氏菌的快速检测方法。
背景技术
在食品安全问题日益多元化的今天，占人类食品中特殊地位的乳及乳制品的安全性一直是社会和科学研究关注的焦点。乳制品营养丰富、搭配均衡，是一种经济实惠的优质蛋白，我国是乳制品消费大国，也是世界第三大乳制品生产国。然而，乳制品极易遭受致病菌污染，常见的污染乳制品的食源性致病菌有沙门氏菌、金黄色葡萄球菌、阪崎肠杆菌等，其进入人体内后会造成腹泻、呕吐、急性肠胃炎等病症，严重的会危及生命。其中沙门氏菌和金黄色葡萄球菌是乳品检测最普遍的项目。
沙门氏菌是生乳及乳制品中比较常见的一种食源性致病菌，受其感染的人和动物会出现伤寒、副伤寒等病症，沙门氏菌一旦进入血液组织，则会导致系统性炎症，甚至死亡。对于一个中等规模的乳品工厂，一天生产的不同类产品至少有上百个批次，加工过程中的原料、过程品、终产品、生产环境都需要进行沙门氏菌检测。根据GB4789.1-2016规定同一批次需采集5个样品进行检测，检测量每天高达几百个。根据GB4789.4-2016中要求，沙门氏菌的定性定判至少需要3天，该方法操作较繁琐、耗时长，不能满足检测时效的需求。
发明内容
针对现有技术的不足之处，本发明的目的在于提供一种乳制品中沙门氏菌的快速检测方法。
本发明的技术方案概述如下：
(1)预处理：将待检测的乳制品加入含有二乙酰胺四乙酸钠和葡萄糖苷 的溶液中，在40-42℃下水浴50-60分钟；
(2)提取待测样品中DNA模板：将步骤(1)中水浴后的混合溶液过滤，并用生理盐水洗脱过滤膜上的细菌，然后离心洗脱液，弃去上清液，向沉淀物中加入1-1.2mL的TE缓冲溶液，震荡使沉淀物悬浮，再次离心并弃去上清液，用细菌基因组DNA提取试剂盒快速提取被检测样品中DNA；
(3)配制LAMP扩增反应液：3μL的10×ThermoPol反应缓冲溶液、1μL浓度为1.6mM的dNTP、2μL浓度为25μM的FIP引物、2μL浓度为25μM的BIP引物、1μL浓度为25μM的F3引物、1μL浓度为25μM的B3引物、0.8μL的Bst聚合酶、8μL的待测样品DNA、2μL浓度为1.2mM的曙红、2μL浓度为1mM的2-羟基-3-萘甲酸、7.2μL灭菌超纯水；
其中，所述FIP引物、BIP引物、F3引物、B3引物分别为沙门氏菌invA基因的LAMP检测引物中的前内引物、后内引物、前外引物、后外引物；
(4)LAMP扩增及检测：将步骤(3)配制的扩增反应液在65℃恒温反应45-50分钟，进行荧光显色检测；当LAMP扩增反应液由褐色变为黄绿色荧光时，具有阳性扩增；当LAMP扩增反应液无颜色变化时，不具有阳性扩增。
优选的是，步骤(1)中所述的溶液中二乙酰胺四乙酸钠的质量浓度为40-50wt％，葡萄糖苷的质量浓度为20-25wt％。
优选的是，步骤(1)中所述乳制品、二乙酰胺四乙酸钠、葡萄糖苷三者的质量比为1∶4-5∶2-2.5。
优选的是，步骤(2)中所述过滤采用无菌操作。
优选的是，步骤(2)中所述的离心为在12000rpm转速下离心5分钟。
优选的是，步骤(3)中所述
FIP引物的序列组成是5’-GCGCCGCTA CGCACTCTA TATGTAGCT GCTACGCAC GG-3’；
BIP引物的序列组成是5’-GAACGGTGA AGCTTACGG GACGTCACT CGGTCATAG GAA-3’；
F3引物的序列组成是5’-GAACATGCT GCAGATGTC-3’；
B3引物的序列组成是5’-CGGCATTAG CGACTCCTT-3’。
优选的是，步骤(4)中所述荧光显色检测采用IQ TN5荧光定量PCR仪光 学系统。
对本发明及其有益效果的阐述：本发明所涉及的对乳制品中沙门氏菌的检测方法简单省时、准确度高、灵敏度高，从对乳制品的预处理到得到最终检测结果能够仅需要3-3.5小时，极大的提高了被检乳制品的新鲜度和货架期，通过本发明所述方法检测出的结果与按照GB4789.4-2016中步骤检测出的结果的阳性符合率达到99.9％以上；首先，本方法中将待检乳制品用含有二乙酰胺四乙酸钠(EDTTI-Na)和葡萄糖苷(APG)的溶液进行了预处理，EDTTI-Na的结构与EDTA近似，用酰胺基取代了EDTA中的胺基，增加了分子量，从而提高了螯合能力，能与钙离子形成稳定的六圆环螯合物，EDTTI-Na在APG的辅助作用下，夺取乳制品酪蛋白胶束中的钙离子、分解体系中的大分子蛋白聚合物，使酪蛋白胶束解离成小分子酪蛋白单体、蛋白聚合物解离成短肽链，克服蛋白胶束或者聚合物在真空快速过滤富集过程中的阻遏作用，从而能够迅速富集所要检测样品，即含有沙门氏菌等的菌体。
然后，在配制LAMP扩增反应液时，(1)以美国基因序列数据库(Gene Bank)中Gene Bank号为M90846的沙门氏菌invA基因作为靶基因，设计出其LAMP扩增的两对最佳引物，即内引物(FIP、BIP)和外引物(F3、B3)，对靶基因进行扩增时，特异性强，能够准确排出其他生理生化相似的肠杆菌科细菌的干扰，对多种不同血清型沙门氏菌均能有效扩增；(2)在反应液中加入了荧光显色反应试剂，曙红和2-羟基-3-萘甲酸，两者在单独使用时没有针对沙门氏菌靶基因扩增产物的特异性荧光显色效果，当按照本发明所述的比例添加时，对沙门氏菌invA基因的阴性及阳性反应具有明显的颜色区分，可据此准确判断结果，即当LAMP扩增反应液由褐色变为黄绿色荧光时，具有阳性扩增，意味着样品中存在沙门氏菌污染；当LAMP扩增反应液无颜色变化时，不具有阳性扩增，说明样品中不存在沙门氏菌污染。
最后，本发明所涉及的荧光检测沙门氏菌的灵敏度高，较常用的PCR电泳检测灵敏度提高了10倍以上，而且操作简便，对靶基因的富集效率高，在半小时的扩增时间中足以达到能够检测的限度。
具体实施方式
下面结合实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。本发明提供了一种乳制品中沙门氏菌的快速检测方法，通过下述实施例具体阐述。
实施例1
(1)预处理：将1重量份的待检测的牛乳加入10重量份的含有50wt％二乙酰胺四乙酸钠和25wt％葡萄糖苷的溶液中，在40-42℃下水浴50-60分钟；
(2)提取待测样品中DNA模板：将步骤(1)中水浴后的混合溶液无菌真空抽滤，并用生理盐水洗脱过滤膜上的细菌，然后以12000rpm离心洗脱液5分钟，弃去上清液，向沉淀物中加入1.2mL的TE缓冲溶液，震荡使沉淀物悬浮，再次离心并弃去上清液，用细菌基因组DNA提取试剂盒快速提取被检测样品中DNA；
(3)配制LAMP扩增反应液：3μL的10×ThermoPol反应缓冲溶液、1μL浓度为1.6mM的dNTP、2μL浓度为25μM的FIP引物、2μL浓度为25μM的BIP引物、1μL浓度为25μM的F3引物、1μL浓度为25μM的B3引物、0.8μL的Bst聚合酶、8μL的待测样品DNA、2μL浓度为1.2mM的曙红、2μL浓度为1mM的2-羟基-3-萘甲酸、7.2μL灭菌超纯水；其中，所述FIP引物、BIP引物、F3引物、B3引物的序列组成如前文所述；
(4)LAMP扩增及检测：将步骤(3)配制的扩增反应液在65℃恒温反应45分钟，用IQ TN5荧光定量PCR仪光学系统进行荧光显色检测；当LAMP扩增反应液由褐色变为黄绿色荧光时，具有阳性扩增，说明样品中存在沙门氏菌；当LAMP扩增反应液无颜色变化时，不具有阳性扩增，说明样品中不存在沙门氏菌。
取相同批次的牛乳样品，分为两部分，第一部分通过实施例1中的方法检测牛乳是否被沙门氏菌污染，在3小时内能够得到结果；同时第二部分按照GB4789.4-2016中步骤进行沙门氏菌检测，最快需要3个工作日能够得到测试结果；在不少于100次的测试中，两种方法对沙门氏菌的测试结果完全相符。当将测试对象从牛乳变为全脂乳粉后，通过本发明的沙门氏菌检测方法的检测时间约3.5小时，在不少于100次的测试中，与通过GB4789.4-2016 测试的结果相同。
实施例2
将实施例1步骤(1)中二乙酰胺四乙酸钠和葡萄糖苷用相同浓度的常规试剂乙二酸四乙酸(EDTA)代替，进行乳制品的预处理，其余检测方法与实施例相同。
在用EDTA替换后，步骤(2)中真空抽滤富集细菌时阻力较大，阻遏作用明显，对等量样品的富集时间增长约25-30分钟。
实施例3——通用性检测
选取3种不同血清型的常见沙门氏菌(肠炎沙门氏菌、伤寒沙门氏菌、猪霍乱沙门氏菌)用煮沸法提取基因组DNA，同时以双蒸水作为阴性空白对照组样品，按照实施例1步骤(3)和步骤(4)中方法进行LAMP扩增并检测。
荧光显色检测结果为5种不同血清型沙门氏菌LAMP反应液均由褐色变为黄绿色荧光，表明均具有阳性扩增；阴性对照反应前后LAMP反应液为褐色，无颜色变化，表明未出现阳性扩增。
当在步骤(3)的LAMP反应液中不加入曙红或者2-羟基-3-萘甲酸时，即使LAMP扩增反应超过1小时，不会出现任何荧光显色的变化。
实施例4——特异性检测
选取与沙门氏菌生理生化特征相似的5种肠杆菌科细菌(沙雷氏菌、变形杆菌、志贺氏菌、大肠杆菌、克雷伯氏菌)用煮沸法提取基因组DNA，同时以沙门氏菌基因组DNA为阳性对照、双蒸水以阴性对照，按照实施例1步骤(3)和步骤(4)中方法进行LAMP扩增并检测。
荧光显色检测结果为只有肠炎沙门氏菌LAMP反应液由褐色变为黄绿色荧光，表明具有阳性扩增，证实了沙门氏菌的存在；其它肠杆菌科细菌以及阴性对照反应前后LAMP反应液均为褐色，无颜色变化，表明未出现阳性扩增，说明不存在沙门氏菌。
实施例5——灵敏度检测
煮沸法提取肠炎沙门氏菌标准菌株基因组DNA，并进行10倍梯度稀释，分别得到浓度为445ng/μL、44.5ng/μL、4.45ng/μL、445pg/μL、44.5pg/μL、4.45pg/μL、0.445pg/μL、0.0445pg/μL的肠炎沙门氏菌基因组DNA。 将不同梯度DNA浓度按照实施例1步骤(3)和步骤(4)中方法进行LAMP扩增并检测。
在经过45分钟的扩增反应后，荧光显色检测结果为445ng/μL—0.445pg/μL的肠炎沙门氏菌LAMP反应液均变为黄绿色荧光，仅有0.0442pg/ul的浓度LAMP反应液未出现颜色变化，为褐色。该检测线的最低限度较现有技术中的检测限度低了超过10倍，对沙门氏菌的灵敏度大大提高。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节。

Claims (7)

  1. 一种乳制品中沙门氏菌的快速检测方法，其特征在于，包括下述步骤：
    (1)预处理：将待检测的乳制品加入含有二乙酰胺四乙酸钠和葡萄糖苷的溶液中，在40-42℃下水浴50-60分钟；
    (2)提取待测样品中DNA模板：将步骤(1)中水浴后的混合溶液过滤，并用生理盐水洗脱过滤膜上的细菌，然后离心洗脱液，弃去上清液，向沉淀物中加入1-1.2mL的TE缓冲溶液，震荡使沉淀物悬浮，再次离心并弃去上清液，用细菌基因组DNA提取试剂盒快速提取被检测样品中DNA；
    (3)配制LAMP扩增反应液：3μL的10×ThermoPol反应缓冲溶液、1μL浓度为1.6mM的dNTP、2μL浓度为25μM的FIP引物、2μL浓度为25μM的BIP引物、1μL浓度为25μM的F3引物、1μL浓度为25μM的B3引物、0.8μL的Bst聚合酶、8μL的待测样品DNA、2μL浓度为1.2mM的曙红、2μL浓度为1mM的2-羟基-3-萘甲酸、7.2μL灭菌超纯水；
    其中，所述FIP引物、BIP引物、F3引物、B3引物分别为沙门氏菌invA基因的LAMP检测引物中的前内引物、后内引物、前外引物、后外引物；
    (4)LAMP扩增及检测：将步骤(3)配制的扩增反应液在65℃恒温反应45-50分钟，进行荧光显色检测；当LAMP扩增反应液由褐色变为黄绿色荧光时，具有阳性扩增；当LAMP扩增反应液无颜色变化时，不具有阳性扩增。
  2. 根据权利要求1所述的检测方法，其特征在于，步骤(1)中所述的溶液中二乙酰胺四乙酸钠的质量浓度为40-50wt％，葡萄糖苷的质量浓度为20-25wt％。
  3. 根据权利要求1所述的制备方法，其特征在于，步骤(1)中所述乳制品、二乙酰胺四乙酸钠、葡萄糖苷三者的质量比为1∶4-5∶2-2.5。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述过滤采用无菌操作。
  5. 根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述的离心为在12000rpm转速下离心5分钟。
  6. 根据权利要求1所述的检测方法，其特征在于，步骤(3)中所述
    FIP引物的序列组成是5’-GCGCCGCTA CGCACTCTA TATGTAGCT GCTACGCAC GG-3’；
    BIP引物的序列组成是5’-GAACGGTGA AGCTTACGG GACGTCACT CGGTCATAG GAA-3’；
    F3引物的序列组成是5’-GAACATGCT GCAGATGTC-3’；
    B3引物的序列组成是5’-CGGCATTAG CGACTCCTT-3’。
  7. 根据权利要求1所述的制备方法，其特征在于，步骤(4)中所述荧光显色检测采用IQ TN5荧光定量PCR仪光学系统。
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